Immunotherapeutic combinations for the treatment of tumours that overexpress gangliosides

ABSTRACT

The present invention is related to the field of immunology and more specifically with immunotherapeutic combinations used for controlling growth and/or cell proliferation of tumors. By means of the present invention a therapeutic effect against tumors is observed by the combination of idiotypic vaccines whose principle active is an anti-ganglioside antibody (Ab1), with idiotypic vaccines whose principle active is anti-idiotype antibody (Ab2) obtained against an anti-ganglioside antibody, or vaccines whose principle active is one or more gangliosides. Combinations of these vaccines are described that cause a synergic therapeutic effect against tumors. The referred combinations can be applied to patients in different clinical conditions of tumors that express gangliósidos.

TECHNICAL FIELD

This invention relates to human medicine, and especially to therapeuticvaccines that induce an immune response to tumors that over-expressgangliosides.

PRIOR ART

Gangliosides are components of the plasmatic membranes of the most ofmammalian cells. Even when these glycosphingolipids are expressed innormal tissues, they are very attractive targets for immunotherapy dueto the expression patterns during the malignant transformation of thecells (Hakomori, S. Ann. Rev. Biochem. 50; 1981:733-764; Hakomori,Cancer Res. 45; 1985:2405-2414; Irie, R. F., et al. In: Therapeuticmonoclonal antibodies. Borrebaeck, C. A. K., and Larrick, J. W. (Eds.).M Stockton Press, 1990, pp. 75-94).

The saccharide nature of the gangliosides, together to the fact thatthey are self-antigens makes them very low immunogenic molecules. Somestrategies have been used to increase the immunogenicity of theseantigens. They are based on presentation of gangliosides to the immunesystem in a different molecular environment. One of these strategies hasbeen the use of conjugated vaccines in which the saccharide antigen iscovalently linked to a carrier protein where the carrier is veryimmunogenic for T cells. It makes possible to obtain a strong and longlasting antibody immune response. Using this procedure has been possiblerise IgG antibodies against such gangliosides, although the titers afterthe re-immunizations were smaller in comparison with those obtained in aclassic immune response against thymus-independent antigens (Helling, Fet al. Cancer Res. 54; 1994:197-203; Helling, F et al. Cancer Res. 55;1995:2783-2788; Livingston P O et al. Cancer Immunol Immunother 45;1997:10-19).

New vaccine compositions have been reported to induce an immune responseagainst N-acetylated and N-glycolylated gangliosides. These vaccines arebased on hydrophobic conjugation between gangliosides and very smallsize proteoliposomes (VSSP) obtained from the association of the OuterMembrane Protein Complex (OMPC) from Neisseria meningitidis,Gram-negative bacteria, with gangliosides (gangliosides/VSSP) (EstévezF. Et al. Vaccine 18; 1999:190-197; U.S. Pat. No. 5,788,985 y U.S. Pat.No. 6,149,921). The vaccines composed by GM3 ganglioside/VSSP or the(NeuGcGM3) ganglioside/VSSP have raised high IgM and IgG antibodiestiters specific against GM3 and NeuGcGM3.

Otherwise, vaccination with GM3/VSSP increased survival of mice bearingmelanoma B16, it also reduced tumor volumes and it increased rejectionrate to the subcutaneous transplant of the tumor (Alonso et al. Int. JOncology 15; 1999: 59-66; Car, A. et al. Melanoma Research, in press).

The idiotype network theory proposed by Jerne 1974 (Jerne, N. K. Ann.Immunol. 125C; 1974:373-389), pointed out the immune system as anantibody network with a complex interaction pattern between them andseveral natural antigens, that interactions happen through the variableregions or idiotype (Id) and by interfacing idiotype-antiidiotype, theimmune system is regulated. Jerne's theory supported new strategies forcancer active immunotherapy (AI) based on the use of anti-idiotypevaccines. The vaccination is carried out with an antibody (Ab1) thatrecognizes the tumor-associated antigen, which induces anti-idiotypeantibodies (Ab2). These anti-idiotype antibodies mimic the nominalantigen and in turn they generate anti-anti-idiotype antibodies (Ab3)against the tumor-associated antigen (Schmolling J, et al. Hybridoma 14;1995:183-186). On the other hand, the induction of immune responseagainst tumor associated antigens has been reported after vaccinationwith anti-Id antibodies (Ab2) (Raychauhuri, S. et al. J. Immunol. 137;1986:1743-1749; Raychauhuri, S. et al. J. Immunol. 139; 1987:3902-3910;Bhattacharya-Chatterjee, M. et al. J. Immunol. 139; 1987:1354-1360;Bhattacharya-Chatterjee, M. et al. J. Immunol. 141; 1988:1398-1403;Herlyn, D. et al. Intern. Rev. Immunol. 4; 1989:347-357; Chen, Z-J. etal. Cell. Imm. Immunother. Cancer; 1990:351-359; Herlyn, D. et al. InVivo 5; 1991:615-624; Furuya, A. et al. Anticancer Res. 12; 1992:27-32;Mittelman, A. et al. Proc. Natl. Acad. Sci. USA 89; 1992:466-470;Durrant, L G. et al. Cancer Res. 54; 1994:4837-4840; Mittelman, A. etal. Cancer Res. 54; 1994:415-421; Schmitt, H. et al. Hybridoma 13;1994:389-396; Chakrobarty, M. et al. J. Immunother. 18; 1995:95-103;Chakrobarty, M. et al. Cancer Res. 55; 1995:1525-1530; Foon, K A. et al.Clin. Cancer Res. 1; 1995:1285-1294; Herlyn, D. et al. Hybridoma 14;1995:159-166; Sclebusch, H. et al. Hybridoma 14; 1995:167-174; Herlyn,D. et al. Cancer Immunol. Immunother. 43; 1996:65-76).

Another choice, concerning the use of Ab2 antibodies and in order toincrease the immunogenicity of saccharide residues, involves thepossibility that such saccharide epitopo can be represented by a proteinepitopo on the antibody molecule. In fact, many of these anti-Idantibodies have been obtained; they mimic gangliosides highly expressedin tumor cells such as GM3, GD3 and GD2 (Yamamoto, S et al. J. Natl.Cancer Inst. 82; 1990:1757-1760; Chapman, P. B. et al. J. Clin. Invest88; 1991:186-192; Cheung N-K V, et al. Int J Cancer 54; 1993:499-505;Saleh M N. Et al. J Immunol 151; 1993:3390-3398; Sen G. Et al. JImmunotherapy 21; 1998: 75-83).

Promissory results have been achieved from clinical trials in cancerpatients by using Ab2 antibodies simultaneously with BCG or QS21 asadjuvant (McCaffery M. Et al. Clin Cancer Res 2; 1996: 679-686.; Foon KA. Et al. Clin Cancer Res. 4; 1998:1117-1124).

The monoclonal antibody P3 (Deposit Number ECACC 94113026) is known fromthe prior art, which specifically recognizes the sialic acid inN-glycolyl-containing monosialo and disialogangliosides. This Mab Ab1recognizes antigens on human breast tumors and melanomas. (U.S. Pat. No.5,788,985; Vázquez A M. Et al. Hybridoma 14; 1995:551-556; Moreno E. Etal. Glycobiology 8; 1998: 695-705; Marquina G. Et al. Cancer Res 56;1996:5165-5171; Carr, A. Et al. Hybridoma 19 (3); 2000:241-247).

The anti-idiotype antibody 1E10 (Ab2 MAb 1E10) (Deposit Number ECACC97112901) obtained by immunization with MAb P3 is a gamma type antibody(not internal image). Ab2 MAb 1E10 showed anti-tumor effect on growthtumors of breast and melanomas (U.S. Pat. No. 6,063,379; Vázquez et al.Hybridoma 14; 1995:183-186; Vazquez et al. Oncology Reports 7;2000:751-756).

Do not exist evidences in the prior art about the use of the combinationbetween vaccines comprising gangliosides and pharmaceutical compositionscomprising anti-gangliosides antibodies (Ab1) or anti-idiotypeantibodies (Ab2), named idiotypic vaccines; neither the use ofcombinations of idiotypic vaccine Ab1 with idiotypic vaccine Ab2.

The present invention refers the use of all the possible vaccinecombinations described, in order to potentiate the effect that theyproduce each one of them separately.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to a pharmaceutical composition or thecombination of pharmaceutical compositions, useful for cancerimmunotherapy, specifically for tumors that over express gangliosides,and that it comprises at least two of the following compounds:

-   -   (A) An idiotypic vaccine comprising an anti-ganglioside antibody        (Ab1);    -   (B) An idiotypic vaccine comprising an anti-idiotype antibody        (Ab2) against an anti-ganglioside antibody; and    -   (C) A vaccine comprising a ganglioside.

The pharmaceutical composition, or the combination of pharmaceuticalcompositions can have A plus B, or A plus C, or B plus C.

Preferably that pharmaceutical composition, or combination of thepharmaceutical compositions, where A can be an idiotypic vaccinecomprising the murine MAb P3 with Deposit Number ECACC 94113026; andwhere B can be an idiotypic vaccine comprising the murine anti-idiotypeantibody 1E10 with Deposit Number ECACC 97112901 and where C can be avaccine comprising N-glycolyl GM3 (NeuGcGM3) or N-acetyl GM3 (NeuAcGM3)gangliosides.

In the present invention A plus B, or A plus C, or B plus C can beadministered in a simultaneous or alternating way.

The compositions of this invention may be useful in the treatment ofcancer, particularly those which over express gangliosides as well aslung, breast, digestive system, urogenital system, melanomas, sarcomasand those derived from neuroectodermic tissue.

The present invention also described the scheme for the administrationof the pharmaceutical compositions, or combination of pharmaceuticalcompositions to treat mammalians.

In the present invention a method is also described a method thatcomprises the administration to mammals of the pharmaceuticalcomposition, or combination of pharmaceutical compositions describedpreviously for the prevention or treatment of tumors of breast, lung,digestive system, urogenital system, melanomas, sarcomas and ofneuroectodermic origin.

1. Preparation of Vaccine Comprising Gangliosides:

The vaccines comprising gangliosides are obtained according to thespecification of Estevez and col in Vaccine 18,1999: 190-197 and in U.S.Pat. No. 5,788,985 y U.S. Pat. No. 6,149,921). Very Small SizeProteoliposomes (VSSP) obtained from the association of the OuterMembrane Protein Complex (OMPC) from the Gram-negative bacteria strain,Neisseda meningitidis, with synthetic or natural gangliosidesincorporated therein (VSSP-G). The gangliosides could be (NeuGcGM3), GD3or (NeuAcGM3).

2. Preparation of Idiotypic Vaccines:

The pharmaceutical compositions are obtained according to thespecifications of U.S. Pat. No. 5,817,513 and U.S. Pat. No. 6,063,379.The vaccine comprises murine anti-ganglioside monoclonal antibodies suchanti-gangliósidos such as MAb P3 or murine anti-idiotype monoclonalantibodies such as Mab 1E10 that recognize anti-ganglioside monoclonalantibodies.

3. Immunotherapeutic Combinations that Potentiate the Immune Responseand the Antitumor Effect of Ganglioside Vaccines and Idiotypic Vaccinesin Animal Models:

The referred procedures can be used in mice or any other species ofmammals. The components of the combination are the ganglioside vaccineand the idiotypic vaccine; they can be combined in different ways.

In one combination, the animals can be immunized with 3 to 10 doses in arange of 25 μg to 1 mg of the murine anti-ganglioside monoclonalantibody; the interval between doses can be 7 and 14 days. During thisperiod the animals receive among 3 to 10 dose in a range among 60 to1000 μg of the ganglioside vaccine, the interval between doses can be 7to 14 days.

In another combination, the animals can be immunized with 3 to 10 dosein a range of 25 μg to 1 mg of the murine anti-idiotypic antibodyspecific against an anti-ganglioside antibody; the interval betweendoses can be 7 to 14 days. During this period the animals receive among3 to 10 dose in a range among 60 to 1000 μg of the ganglioside vaccine,the interval between doses can be 7 to 14 days.

The administration of both types of vaccines can be simultaneous oralternating. The vaccines can be formulated as separate products or as avaccine composition when they are administered in a simultaneous way.When vaccines are administered in an alternating way, the intervalsbetween each type of vaccine can be 3-7 days. The vaccines areadministered in an adjuvant that can be aluminum hydroxide (62.5 μg-2.5mg), Montanide ISA 51 (0.1-1.2 ml/doses), or any other appropriateadjuvant. The total volume for each dose can be 10 μl-2 ml. Vaccinescomprising gangliosides can be administered intradermic, subcutaneous,intramuscular, intraperitoneal, intramucosal or their combinations. Thesame administration routes can be used for vaccines comprisingantibodies.

The immunotherapeutic combinations increase the antibody immune responseas well as the cellular immune response in treated animals.

The time of appearance of local tumors, the tumor volume and survival oftreated subjects is compared with the same parameters for the controlgroup, in order to evaluate the effectiveness of immunotherapeuticcombinations. When comparing these parameters among treated and controlgroups it can be observed a shorter time of appearance of local tumors,a decrease of the tumor volume and an increase in survival for the grouptreated with therapeutic combinations of the present invention. Thecontrol groups are not only those animals treated with the adjuvant, butalso those groups treated with just only one vaccine instead of withtheir combinations.

4. Immunotherapeutic Combinations that Potentiate the Immune Responseand the Antitumor Effect of Ganglioside Vaccines and Idiotypic Vaccinesin Human Patients:

The procedure before referred can also be applied to cancer patients indifferent clinical stages. Particularly those tumors that over expressgangliosides as well as lung, breast, digestive system, urogenitalsystem, melanomas, sarcomas and those derived from neuroectodermictissue

In one combination, the patients can be immunized with 3 to 10 dose in arange of 0.1 to 5 mg of the murine anti-ganglioside monoclonal antibody;the interval between doses can be 7 and 14 days. During this period thepatients receive among 3 to 10 dose in a range among 60 to 1000 μg ofthe ganglioside vaccine, the interval between doses can be 7 to 14 days.

In a second combination, the patients can be immunized with 3 to 10 dosein a range of 0.1 to 5 mg of the murine anti-idiotypic antibody specificagainst an anti-ganglioside antibody; the interval between doses can be7 to 14 days. During this period the patients receive among 4 to 6 dosein a range among 60 to 1000 μg of the ganglioside vaccine, the intervalbetween doses can be 7 to 14 days.

In a third combination, the patients can be immunized with the murineanti-ganglioside monoclonal antibody; doses, frequency and intervals canbe the described previously. During this period the patients receiveamong 3 to 10 dose in a range among 0.1 to 2 mg of the murineanti-idiotypic antibody specific against an anti-ganglioside antibody.

The administration of both types of vaccines can be simultaneous oralternating. The vaccines can be formulated as separate products or as avaccine composition when they are administered in a simultaneously. Whenvaccines are administered in an alternating way, the intervals betweeneach type of vaccine can be 3-7 days. The vaccines are administered inan adjuvant that can be aluminum hydroxide (1-5 mg/doses), Montanide ISA51 (0.6-1.2 ml/doses), or any other appropriate adjuvant. The totalvolume for each dose can be 10 μl-2 ml.

Vaccines comprising gangliosides can be administered intradermic,subcutaneous, intramuscular, intraperitoneal, intramucosal or theircombinations. The same route can be used for vaccines comprisingantibodies.

During vaccination, some biochemical parameters and antibodies titersare monitored by measurements in blood. The frequency can be 1 week to 3months. Cellular immunity is studied using lymphocytes from thepatients. The extractions are carried out with a frequency oscillatingfrom one week to three months.

Finally, the patients are re-immunized with both vaccines at theconcentrations before mentioned; the intervals between doses can be 1-6months. They can be administered simultaneously or not and during 1 to 2years.

The present vaccination scheme induces in patients an antibody andcellular immune response increased, and therefore to reduce tumorburden.

EXAMPLES Example 1 Activation of Anti-Idiotype T2 and Anti-Anti-Idiotype T3 Cells in the Syngeneic Model, Induced by Immunizationwith Murine MAB P3

Female Balb/c and nude mice of the same genetic background 6-8 weeksold, were immunized subcutaneously with 100 μg of murine MAb P3(anti-N-glycolylated ganglioside, Deposit Number ECACC 94113026; Vázquezet al., Hybridoma 14; 1995: 551-558; Moreno et al. Glycobiolgy 8; 1998:695-705) and complete Freund's adjuvant (CFA). Seven days later micewere re-immunized with 50 μg of the antibody in incomplete Freund'sadjuvant (IFA). At day 10, draining lymph nodes were collected and thelymphocytes were obtained by pressing with a syringe. The cellularsuspension was used in experiments of cellular proliferation, which wasmeasured by ³H-thymidine incorporation. In vitro lymphocyteproliferation was measured by cultivating lymphocyte suspension in thepresence of increased concentrations (25 to 150 μg/mL) of the murine MAbP3 and its Ab2 MAb 1E10 (Number of Deposit ECACC 97112901).Isotype-matched murine MAbs were used as controls. Stimulation indexesequal to or higher than 3 where considered positive. Lymphocytesobtained after the immunization of Balb/c mice with P3 MAb specificallyproliferated in the presence of this MAb and this proliferation was notobserved when the cells were cultured in the presence of the murinecontrol MAb A3 (IgM) (Alfonso M. and col.: Hybridoma 14; 1995: 209-216).This proliferation was dependent on the presence of T cells; because itwas not obtained with lymph node cells from P3 immunized athymic nu/numice. Immunization with the murine MAb P3 not only induced theproliferation of T cells against the idiotype of this Ab1 MAb (T2), butalso induced the proliferation of anti-anti-idiotype T cells (T3)specific for the murine Ab2 MAb 1E10 (IgG1), and not against Mab C5 ofthe same isotype (IgG1) (FIG. 1).

Example 2 Activation of Anti-Idiotype (T2) and Anti-Anti-Idiotype (T3)Induced by Immunization with the Chimeric Antibody P3

Chimeric antibody P3, whose amino acid sequences of its variable regionsof the heavy (VH) and light (VL) chains are respectively shown in FIGS.8 and 9, was used to immunize female Balb/c mice, 6-8 weeks old. For thefirst subcutaneous dose, 100 μg of the antibody emulsified in ACF wasused. Seven days later animals were re-immunized with 50 μg of theantibody emulsified in AIF. At day 10, draining lymph nodes (LN) werecollected and the LN cell lymphoproliferative capacity was analyzed byincubating in the presence of the murine and chimeric variants ofantibodies P3 and Ab2 1E10, whose sequences of the variable regions areshown in the FIGS. 13 (heavy chain) and 14 (light chain). C5 murine MAband its chimeric variant were used as isotype-matched controls (PatentApplication WO 97/33916 A1). Lymphocytes from mice immunized with thechimeric antibody P3 specifically proliferated when cultivated in thepresence of this antibody or the chimeric 1E10 MAb, and also whencultivated in the presence of the murine variants of these antibodies.Specificity of proliferation was demonstrated by using isotype-matchedcontrol MAbs. Therefore, chimeric P3 MAb maintains the ability of themurine variant to induce specific proliferation of anti-idiotype (T2)and anti-anti-idiotype (T3) T cells (FIG. 2).

Example 3 Reinforcement of the Anti-Tumor Effect of theGM3-VSSP/Montanide ISA 51 Vaccine by its Combination with a VaccineComposed by the Murine MAb 1E10 Adsorbed in Aluminum Hydroxide

A group of 6-8 weeks old C57BL/6 female mice was immunizedsubcutaneously at days 0, 14, 28 and 42 with 50 μg of murine 1E10 MAbadsorbed in aluminum hydroxide. At days 7, 21, 35 and 49, mice receivedintramuscular doses with 120 μg of GM3-VSSP/Montanide ISA 51. Anothergroup of mice was immunized in a similar way, but beginning theimmunizations with the vaccine preparation containing GM3 ganglioside.Control groups separately immunized at the same time intervals and withthe same number of doses of each vaccine or only with phosphate-bufferedsaline solution were used in these experiments. At day 63, mice in allgroups were subcutaneously inoculated with 10 000 cells of the murinetumor B16. Tumor growth was evaluated in all groups.

Mixed Lineal Pattern statistical data analysis was used for comparisonof tumor growth between all groups and the group control that onlyreceived PBS. Vaccination with 1E10 MAb alone didn't protect miceagainst a challenge with 10,000 melanoma B16 cells. VSSP-GM3 vaccinecaused a retard of the tumor growth (p<0.05). The combinations ofGM3-VSSP and 1E10 MAb vaccines led to an increase in the protectiveeffect observed with GM3-VSSP vaccine alone (p<0.05) (FIG. 3).

Example 4 Immunization Schedule of Cancer Patients with NeuGc-GM3/VSSPGanglioside Vaccine Using Montanide ISA 51 as Adjuvant

Aiming to demonstrate the safety and immunogenicity of the immunizationwith the NeuGc-GM3/VSSP vaccine using Montanide ISA 51 as adjuvant (U.S.Pat. No. 5,788,985 and U.S. Pat. No. 6,149,921), a clinical trial wascarried out in which 20 patients with advanced malignant melanoma, whichwere not eligible for any other onco-specific treatment, were immunizedwith the vaccine.

The patients received 9 doses of the vaccine preparation containing 200μg of GM3 ganglioside. Doses were administered at days 0, 14, 28, 42,56, 84, 112, 140, and 168. According to physician's criteria, patientsreceived additional doses every 28 days after the sixth month until theycompleted one year of treatment.

Blood samples were obtained for routine biochemistry determinations andfor evaluating serum titers of anti-NeuGcGM3 ganglioside antibodies atdays 0, 14, 28, 56, 112, 168, 224, 280, and 332.

Antibodies titers were measured by ELISA. Serum dilutions wereconsidered positive when anti-ganglioside OD values were equal or higherthan 0.1 (referred to anti-methanol OD).

The toxicity of the vaccine NeuGcGM3-VSSP/Montanide ISA 51 consisted onerythema, local pain, induration in the place of injection and fever,and this allowed classification of the toxicity as mild, grade I/IIaccording to the OMS criteria.

Patients developed specific antibody titers against NeuGcGM3 (between1:80 and 1:2560). Detected antibody isotype included IgG and IgM (allpatients) and IgG (75% of the patients; Table I).

In Patient 01, which entered into the trial with a diagnosis of advancedmalignant melanoma (Evolutionary Metastatic Disease, EMD), regressionand stabilization of some cutaneous lesions was observed after twomonths of treatment, with uncolored halos around these lesions in whichthe presence of inflammatory infiltrate and necrosis was demonstrated byanatomopathologic studies in biopsies (FIG 4.).

In patient 02, with diagnosis of advanced malignant melanoma, astabilization of lung metastases lesion was observed in the right vertex18-20 mm, during at least 4 months (FIG. 5). TABLE I Antibody titersagainst NeuGcGM3 in patients with advanced malignant melanoma immunizedwith the NeuGcGM3/VSSP/Montanide ISA 51 vaccine. Patients Day IgM IgGIgA 01 0 0 0 0 14 640 80 320 28 320 160 320 56 1280 160 320 02 0 160 160160 14 160 160 320 28 160 160 320 56 1280 320 320 03 0 80 320 0 14 320320 0 28 640 320 0 56 640 640 0 04 0 0 0 0 14 160 80 160 28 1280 80 16056 2560 640 2560

Example 5 Immunization Schedule in Cancer Patients with a IdiotypicVaccine Containing 1E10 Ab2 Mab Aluminum Hydroxide-Precipitated

Aiming to demonstrate the safety and immunogenicity of an idiotypevaccine containing the murine anti-idiotype MAb 1E10 (U.S. Pat. No.5,817,513 and U.S. Pat. No. 6,063,379) and aluminum hydroxide asadjuvant, a clinical trial was carried out with 20 advanced malignantmelanoma patients, which were not eligible for any other onco-specifictreatment.

Patients received 4 doses of the vaccine, consisting in 2 mg of the 1E10MAb. Blood samples were obtained before the treatment and 14 days aftereach immunization for routine biochemistry determinations and forevaluating serum titers of antibodies against 1E10 MAb idiotype andNeuGc-GM3 ganglioside. Antibody titers were measured by an ELISA assayconsidering positive values those equal or higher than 0.15.

Vaccine administration to the patients produced mild toxicity,classified as degree I and II, according to the OMS.

In 16 of the 17 valuable patients strong IgG Ab3 antibody responses wereelicited, detectable after receiving 2 or 3 doses of the vaccine. Theanalysis of the specificity of this Ab3, showed a preferentialrecognition by patient's sera of 1E10 MAb, when compared with otherisotype-matched control MAbs, which suggested the induction ofidiotype-specific component in the antibody response to 1E10 MAb. Thiswas corroborated by the strong sera reactivity against the F(ab′)2fragments of this MAb, with a median titer of 1:15000 (titers from1:10000 to more than 1:100000), with little or no recognition of theF(ab′)2 fragments of the control MAbs used as controls.(FIG. 6).

The antibodies generated against the NeuGcGM3 were, in most of thecases, both IgM and IgG, with titers 1:4000 and 1:800, respectively(FIG. 7).

A patient with diagnosis of malignant melanoma and liver metastases wasincluded in the clinical trial; after the treatment it was shownstabilization of the disease for 8 months and the patient's survival was15 months.

Example 6 Combined Immunization with Anti-Idiotypic Vaccine ComprisingAb2 MAb1E10 and NeuGcGM3-VSSP Ganglioside Vaccine

A melanoma patient with metastases, which had been submitted to monthlysurgery, after the diagnosis, received 6 doses of the idiotypic vaccinecontaining Ab2 MAb1E10 and aluminum hydroxide gel (2 mg of the MAb perdose), days 0,14,28,42,56. During this period, also it was administeredto the patient the ganglioside vaccine containing 200 ug NeuGcGM3-VSSPganglioside and Montanide ISA 51 as adjuvant, days 7, 21, 35, 49, 63.After the patient received this immunization scheme, he was re-immunizedmonthly with both vaccines simultaneously during two month. For theperiod of vaccination new lesions didn't appear and the patientcondition was good.

Example 7 Recognition of Tumor Tissues by the MAb 14F7

NeuGcGM3 ganglioside is recognized by MAb 14F7 (patent application WO99/40119). Recognition of tumor tissues by the antibody is shown.

Formalin fixed tissue was included in paraffin. The histology wasevaluated in hematoxillin-eosin colored sections.

These sections were immune staining with the complexbiotin-estreptavidin-peroxidase (Hsu, S. M. y Raine, L. 1981,J.Histochem Cytochem., 29:1349-1353). Briefly, the paraffin was removedand wet sections were treated with H₂O₂ 3% (in methanol solution) for 30minutes, to reduce endogenous peroxidase activity. After incubation withMAb 14F7 (pure) for 1 hour at room temperature, biotinylated anti-mouseimmunoglobulins and streptavidin-peroxidase complex were added(Dakopatts) at room temperature; between incubation times, the sectionswere washed with a Tris-HCl buffer solution. The reaction (POD) it wasdeveloped with 5 mL of Tris-HCl, 0.005 mL buffer solution containingH₂O₂ to 30% and of 3-3 diaminobencidine 3 mg.

After washing with hematoxillin-contrasted water (Mayer), the sectionswere covered with balm and covered. The enzyme reaction renders a browncolor.

The fresh biopsies from pathological tissues were obtained one hourafter surgery. All the tissues were washed with saline solution, andfrozen in liquid nitrogen immediately and they were conserved frozen at−80° C.

The frozen fragments were cut in a cryostat Leica at −25° C. Serialsections with 5 um were obtained, air dried and used immediately orconserved at −20° C. enveloped in aluminum paper; afterward the sectionswere fixed with para-formaldehyde 4% for 20 minutes.

In Table II it is shown the immunostaining with MAb 14F7 in severalhuman tumors. A strong membrane and cytoplasm staining is appreciated inmore than 50% of tumor cells. The dye was very intense in coloncarcinomas, uterus, ovary, sarcomas, lymph nodes and brain metastases ofbreast cancer, as well as melanoma metastases. TABLE II TumorImmunostaining with MAb 14F7. Immunostaining Tumor (Positives/Total)Colon Carcinomas 9/9* Uterus Carcinomas 2/2* Ovary Carcinomas 2/2*Sarcomas 2/2* Breast Tumors and lymph 6/6* node metastases Melanomametastases 2/2**More than 50% of cells showed membrane and cytoplasm intenselyimmunostained.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Mice Balb/c euthymic and athymic mice were immunizedsubcutaneously with 100 μg of the murine MAb P3 in CFA followed by are-immunization with 50 μg of the MAb emulsified in IFA. After threedays, lymph nodes cells were collected and it was carried out theproliferation assay with different concentrations of the MAbs P3, A3,1E10 and C5.

FIG. 2. Mice Balb/c were immunized subcutaneously with 100 pg of thechimeric antibody P3 in CFA followed by a re-immunization with 50 μg ofthe antibody emulsified in IFA. After three days, lymph nodes cells werecollected and it was carried out the proliferation assay with differentconcentrations using murine and chimeric antibodies as control.

FIG. 3. Kinetics of growth for murine B16 tumor in mice immunized withthe immunotherapeutic combinations, specifically the idiotypic vaccinecomprising the Ab2 MAb1E10 and of the GM3-VSSP ganglioside vaccine, asit is detailed in the Example 3.

FIG. 4: Evolution of skin metastases in melanoma patient. The pictureswere taken previous to the immunization with the vaccineNeuGcGM3/VSSP/ISA 51, 2 and 4 months after the treatment. Uncoloredhalos around some lesions are observed, stabilized lesions, in onelesion a diminished size was observed and in one lesion increment wasdetected.

FIG. 5: Evolution of lung metastases in a melanoma patient. The lungright vertex observed by computer assisted axial tomography, the lesionsize was 18×20 mm before the immunization with the vaccineNeuGcGM3/VSSP/ISA 51 and 4 months after the immunization stabilizationof the lesion is observed.

FIG. 6: Recognition of F(ab′)2 fragments from Ab2 MAb1E10 and from otherMAbs with the same isotype by serum from patients immunized with theidiotypic vaccine containing Ab2 MAb1E10 and aluminum hydroxide gel asadjuvant.

FIG. 7: Recognition of GM3(NeuGc) and GM3(NeuAc) gangliosides serum frompatients immunized with the idiotypic vaccine containing Ab2 MAb1E10 andaluminum hydroxide gel as adjuvant.

1. An immunotherapeutic combination for the immunotherapy of tumors thatover-express gangliosides, comprising at least two of the followingcomponents: (A) An idiotypic vaccine comprising a murineanti-ganglioside monoclonal antibody (Ab1); (B) An idiotypic vaccinecomprising an specific murine anti-idiotype monoclonal antibody (Ab2),against an anti-ganglioside monoclonal antibody; (C) A gangliosidevaccine.
 2. An immunotherapeutic combination according to claim 1,wherein said combination comprises A plus B or A plus C or B plus C. 3.An immunotherapeutic combination according to claim 1, wherein saidvaccine A comprises the murine anti-ganglioside MAb P3 (Deposit NumberECACC 94113026).
 4. An immunotherapeutic combination according to claim1, wherein said vaccine B comprises the murine anti-idiotype antibody1E10 (Deposit Number ECACC 97112901).
 5. An immunotherapeuticcombination according to claim 1, wherein said vaccine C comprises theNeuGcGM3 ganglioside.
 6. Immunotherapeutic combination according toclaim 1, wherein said vaccine C comprises the NeuAcGM3 ganglioside. 7.Immunotherapeutic combination according to claims 1, wherein A and B orA and C or B and C are administered simultaneously or alternately. 8.Use of any of the immunotherapeutic combinations of the claim 1 for thetreatment of tumors that over-express gangliosides.
 9. Use according toclaim 8, for treatment of tumor of breast, lung, digestive system,urogenital system, melanomas, sarcomas and those from neuroectodermictissues.
 10. A method for controlling growth and/or cell proliferationof tumors that over-express gangliosides, comprising the administrationto a mammal of an immunotherapeutic combination of claim
 7. 11. Methodaccording to claim 10, wherein said mammals are human.